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AG-2020-176 ORIGINAL ARTICLE doi.org/10.1590/S0004-2803.202100000-29

Assessment of the intestinal microbiota in adults with erosive esophagitis

Diego Cardoso BAIMA, Nayara Salgado CARVALHO, Ricardo Correa BARBUTI and Tomas NAVARRO-RODRIGUEZ

Received: 20 August 2020 Accepted: 7 December 2020

ABSTRACT – Background – The intestinal microbiota influences the appropriate function of the . Intestinal dysbiosis may be asso- ciated with a higher risk of esophageal lesions, mainly due to changes in gastroesophageal motility patterns, elevation of intra-abdominal pressure, and increased frequency of transient relaxation of the lower esophageal sphincter. Objective – The aim of this study was to evaluate the intestinal microbiota in individuals with erosive esophagitis and in healthy individuals using metagenomics. Methods – A total of 22 fecal samples from adults aged between 18 and 60 years were included. Eleven individuals had esophagitis (eight men and three women) and 11 were healthy controls (10 men and one woman). The individuals were instructed to collect and store fecal material into a tube containing guanidine solution. The DNA of the mi- crobiota was extracted from each fecal samples and PCR amplification was performed using primers for the V4 region of the 16S rRNA gene. The amplicons were sequenced using the Ion Torrent PGM platform and the data were analyzed using the QIIME™ software version 1.8. Statistical analyses were performed using the Mann-Whitney non-parametric test and the ANOSIM non-parametric method based on distance matrix. Results – The alpha-diversity and beta-diversity indices were similar between the two groups, without statistically significant differences. There was no statistically significant difference in the phylum level. However, a statistically significant difference was observed in the abundance of the familyClostridiaceae (0.3% vs 2.0%, P=0.032) and in the genus Faecaliumbacterium (10.5% vs 4.5%, P=0.045) between healthy controls and esophagitis patients. Conclu- sion – The findings suggest that reduced abundance of the genusFaecaliumbacterium and greater abundance of the family Clostridiaceae may be risk factors for the development of erosive esophagitis. Intervention in the composition of the intestinal microbiota should be considered as an adjunct to current therapeutic strategies for this clinical condition. Keywords – Erosive esophagitis; intestinal microbiota; metagenomic; Faecalibacterium; Clostridiaceae.

INTRODUCTION important to adequately identify the risk factors associated with these conditions to initiate more effective preventive measures and The human intestinal microbiota has recently become the reduce health care costs. subject of extensive research and knowledge about the resident Dysbiosis influences sensory and motor mechanisms of the species and their influence is growing rapidly. The human diges- upper digestive tract(11-13). This imbalance in the microbiota can tive system houses a complex community of microbial cells that increase the production of intraluminal gases, leading to gastric influence human physiology, metabolism, nutrition and immune distension, increased intra-abdominal pressure, and an increase function(1-4). The imbalance of this microbiota, which is termed in the frequency of transient lower esophageal sphincter (LES) dysbiosis, may be involved in the pathogenesis of various digestive relaxations(14-17). A higher exposure of the esophageal epithelium and extra-digestive diseases that include , to reflux of gastric and duodenal material occurs, which increases inflammatory bowel disease, celiac disease, , gastric the risks of erosive esophagitis and Barrett’s (18,19). cancer, obesity, asthma, diabetes mellitus, coronary disease, atopy, The impact of intestinal dysbiosis in the upper gastrointestinal autism, autoimmune diseases, and others(5-7). tract is still unclear. The objective of our study was to evaluate The frequency of esophageal pathologies has increased in recent and compare the intestinal microbiota in patients with erosive decades. Although the majority of solid organ tumors has decreased esophagitis and in healthy volunteers. This comparison involved a in the last 40 years, esophageal adenocarcinoma (EAC) has become detailed taxonomic description using 16s ribosomal RNA (rRNA) more prevalent over time(8). This increase in the number of EAC metagenomic analysis. cases has been especially apparent in western countries and Asia. In the United States, the incidence of EAC is increasing faster than METHODS any other cancer(9). In addition, the prevalence of gastroesophageal reflux disease (GERD) in North America, Europe, and Southeast Approval Asia increased by approximately 50% in relation to the baseline This study was approved by the Research Ethics Committee of in the early and mid-1990s, and subsequently stabilized(10). It is the Clinical Hospital of the Faculty of Medicine, University of São

Declared conflict of interest of all authors: none Disclosure of funding: no funding received Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brasil. Corresponding author: Diego Cardoso Baima. E-mail: [email protected]

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Paulo (HC-FMUSP; Approval No. 1.463.131). Every patient signed calcium channel blockers, nitrates, anticholinergics, and estrogens; an Informed Consent Form prior to the collection of samples. The bulky hiatal ≥5 cm; long-distance journeys in the last 3 study was conducted with patients registered at the HC-FMUSP. months outside the southeastern region of the country; pregnancy The experimental analysis was carried out at the Laboratory of or breastfeeding patients; previous history of surgery of the up- Medical Research – LIM 46, sector of Parasitology of the Institute per GI tract; obesity defined as a body mass index ≥30 kg/m2; and of Tropical Medicine of the University of São Paulo. consumptive syndrome or malnutrition.

Fecal samples Sample collection and DNA extraction Fecal samples were collected from 22 Brazilian male and female Fecal samples were collected by the participants, who were in- adults aged between 24 and 55 years, from March 2017 to Febru- structed to store the stool in a sterile falcon tube containing 12 mL ary 2018. Of the 22 individuals, 11 had been diagnosed as erosive of guanidine 6M/EDTA 200 nM solution to maintain the integrity esophagitis (EE) (mean age 38.8 years, eight men and three women) of the genetic material of the samples. Samples were immediately and 11 were healthy adults (mean age 34.9 years, delivered to the laboratory, where they were stored at -20°C until 10 men and one woman). Body mass index ranged from 22 to 28 DNA extraction(22,23). The extraction and purification of microbial kg/ m2. Further details of the participants is provided in TABLE 1. DNA was carried out in the Parasitology sector of the Institute of Tropical Medicine, University of São Paulo. Accordingly, 0.25 g of TABLE 1. Sociodemographic data of the study participants. each fecal sample was processed with the DNA Power Soil™ kit Erosive (QIAGEN, Carlsbad, CA, USA) according to the manufacturer’s esophagitis Control instructions.

Sex Microbiome analysis Female 27.3% 9.1% The microbiome was characterized by amplifying the V4 do- main of the bacterial ribosomal 16S segment using the primers F515 Male 72.7% 90.9% (5’-CACGGTCGKCGGCGCCATT-3’) and R806 (5’-GGAC- TACHVGGGTWTCTAAT-3’). The bacterial amplicons were Age (years) 39 (±11) 34 (±6) sequenced using the Ion PGM Torrent™ platform (Invitrogen). Weight (kg) 73 (±6) 74 (±8) The readings obtained after sequencing were processed using QIIME™ version 1.8 pipeline and assigned to taxonomic units. Height (meters) 1.7 (±0.08) 1.7 (0.06) Alpha-diversity (taxonomic diversity within the same population)

2 analysis was done using the Shannon, Simpson, Chao1 indices and Body mass index (kg/m ) 25.5 (±1.9) 24.6 (±1.8) number of species observed. To measure beta-diversity (diversity Data are provided as a percentage (95% confidence interval) or mean (± standard deviation) between populations), principal coordinate analysis (PCoA) was performed based on the UniFrac distance matrix, to demonstrate Examinations similarities or dissimilarities between the samples analyzed. All This study recruited patients with dyspeptic symptoms (heart- sequencing raw reads have been deposited in the National Center burn, epigastric pain, fullness, bloating) and asymptomatic controls. for Biotechnology Information (NCBI) under the project accession The symptomatic patients performed an upper gastrointestinal en- number PRJNA656138. doscopy. Those who presented erosive esophagitis were included in this study and collected stool samples. To assess erosive esophagitis, Statistical analyses we used Los Angeles Classification(20). During the examination, To determine the statistically significant differences in the mi- of the gastric body and antrum were performed, using crobial populations that occurred between the two groups studied, the Operative Link for Gastric Assessment system(21) to rule out rarefaction was first performed for the same number of sequences severe or extensive gastric atrophy. The control group consisted of between all samples (59,300 when comparing the EE patients and asymptomatic volunteers who collected feces for the same analysis. the healthy volunteers). After rarefaction, the relative abundance All participants underwent an anamnesis, physical examination, was calculated using the Mann-Whitney non-parametric test. The and anthropometry examinations. statistical analysis of alpha-diversity was performed using the Mann-Whitney U test. The beta-diversity analysis was performed Inclusion and exclusion criteria using the ANOSIM test, a non-parametric method based on a The male and female patients were 18 to 60 years of age with distance matrix, using the QIIMETM software, with P<0.05 confirmed diagnosis of erosive esophagitis (Los Angeles grades considered statistically significant. Boxplot graphs and tables were A and B) through clinical and endoscopic criteria. The exclusion generated to study the diversity according to Simpson, Shannon, criteria included usage of proton pump inhibitors (PPIs), H2 an- Chao1, and Observed Species values. Bar graphs and tables were tagonists, or antacids in the prior 30 days; usage of antimicrobials generated with means, standard deviation (SD), median, and the or probiotics in the preceding 3 months; presence of histologically 25th (P25) and 75th (P75) percentiles for the taxonomic organization confirmed severe and/or extensive atrophic ; comorbidi- (phylum, class, order, family, and genus). The Kolmogorov-Smirnov ties that may interfere in the motility of the gastrointestinal tract, test was used to assess the normality of the variables analyzed. All including diabetes mellitus, previous cerebrovascular accident, tests performed took into account a bidirectional α of 0.05 and a neurological diseases, autoimmune diseases, , hyperpar- 95% confidence interval, and were performed with SPSS 25 software athyroidism, and mastocytosis; usage of drugs that can interfere (IBM, Armonk, NY, USA) and Excel 2010® software (Microsoft, in the motility of the gastrointestinal tract or in salivation, such as Redmond, WA, USA).

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RESULTS

There were no statistical differences in gender, age, or BMI between the two groups (P>0.05 for all). The severity of the erosive esophagitis assessed was grade A 54.5% (6/11) and grande B 45.5% (5/11). A rarefaction curve was generated to determine whether all operational taxonomic units (OTUs) in the data sets were suf- ficiently evaluated. Each rarefaction curve showed a similar pattern, reaching a plateau and a saturation stage, which indicated that the majority of species present in each sample of the two groups were observed (FIGURE 1). The basis of the rarefaction process was the sample with the fewest sequences (n=59,300). The sequences were FIGURE 2. Boxplot graphs of Alpha-diversity indices. No statistically grouped into OTUs based on 97% similarity using the QIIME™ significant difference in alpha-diversity was evident between the erosive program, with the Greengenes database (version 13.8) as a source. esophagitis (EE) and the control groups. Only groups with an average frequency >0.1% were analyzed.

FIGURE 1. Rarefaction curve showing the estimated number of opera- tional taxonomic units (OTUs) in the control group (blue) and the erosive esophagitis group (red), as a function of the sampling sequencing generated using QIIME™ software.

The Shannon and Simpson Diversity indices, the estimated richness by Chao1, and number of species observed indicate no difference in the alpha-diversity in the two groups (TABLE 2, FIGURE 2). There was no difference in the beta-diversity analyzed using PCoA, based on the weighted and unweighted UniFrac distance matrix (FIGURE 3). No differences were found in the phylum level when the two groups were compared. However, at the genus level, a statistically significant difference was observed in the abundance FIGURE 3. Unifrac analysis of the bacterial community, in erosive of the genus Faecaliumbacterium between healthy controls and EE esophagitis (red) and controls (blue), by the unweighted (A) and patients (FIGURE 4; 10.5% versus 4.5%, P=0.045, FIGURE 5). weighted (B) principal coordinates analysis (PCoA) method. A P-value (ANOSIM) =0.870; B P-value (ANOSIM) =0.430. TABLE 2. Alpha-diversity index. Erosive esophagitis Control Mean SD Median P25 P75 Mean SD Median P25 P75

Simpson 0.93 0.03 0.93 0.9 0.94 0.91 0.09 0.94 0.92 0.97 Chao1 368.41 112.14 342.97 276 515.17 350.57 98.9 384.16 265 421.49 Observed species 330.18 100.73 310 240 456 319.82 89.15 351 243 389

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FIGURE 4. Relative abundance and taxonomic classification, at the level FIGURE 7. Boxplot graph with relative abundance of the family of genus, in fecal samples obtained from patients in the control group and Clostridiaceae from the control group and the erosive esophagitis (EE) group. the erosive esophagitis (EE). DISCUSSION

Erosive esophagitis onset depends on many factors, such as the anti-reflux barrier (lower esophageal sphincter and intact crural diaphragm), adequate esophageal clearance (action of gravity, , and salivation), esophageal mucosa resistance, and intragastric emptying and pressure(24). In addition, some authors observed a higher frequency of bacterial overgrowth of the in patients with es- ophagitis(25). Other study observed that the colonic fermentation of non-digestible carbohydrates by intestinal microbiota caused a higher rate of transient relaxation of the LES, a larger number of episodes of acid reflux, and symptoms of GERD(17). Conversely, others found higher levels of gases in the and (26) FIGURE 5. Boxplot graph with relative abundance of the genus of patients with reflux esophagitis . Bacterial dysbiosis can lead Faecalibacterium in the control group and the esophagitis group (EE). to deconjugation of bile acids, which may have implications on the pathophysiology of gastroesophageal mucosal lesions(27). Finally, At the family level, there was a statistically significant difference a recent study reported the benefit of probiotics along with PPI in in the abundance of the family Clostridiaceae between healthy the treatment of reflux esophagitis, with a reduction of relapses(28). controls and EE patients (FIGURE 6; 0.3% versus 2.0%, P=0.032, The collective findings indicate that alterations of the intestinal FIGURE 7). microbiota are related to the increased production of intraluminal gases and a greater risk of developing esophageal lesions. However, the association between intestinal dysbiosis and es- ophageal involvement is poorly documented. Thus, we performed this study to evaluate the intestinal microbiota in EE patients and healthy individuals. During the selection of participants, we excluded several situations that may influence the composition of the intestinal microbiome. These included recent use of PPIs, antibiotics, or probiotics; severe or extensive ; GI tract surgeries, comorbidities, or medications that interfere with the motility of the GI tract; and recent long-distance journeys, among others. The latter criterion reflected the recent reports that the intestinal microbiome is sensitive to changes in climate and diet(29-32). These exclusion criteria were rigorously applied to reduce possible bias. Our results agree with the literature, revealing a greater abun- dance of four phyla in the intestinal microbiota of patients in both groups (GERD and normal individuals). More than 50 phyla have been identified in the environment. However, the characterization FIGURE 6. Relative abundance and taxonomic classification of bacteria of the human microbiota identifies only four as dominant phyla – at the family level, in fecal samples obtained from healthy controls and Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria(33). We erosive esophagitis (EE) patients. observed no difference in beta-diversity between the two groups,

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without any clusters in the PCoA data (FIGURE 3). There was Another study observed a reduction in the abundance of Faecali- also no difference in alpha-diversity (FIGURE 2). Thus, consider- bacterium in patients with prolonged use of PPI(45), with a lower the ing global biodiversity, the two groups exhibited similar intestinal abundance of Faecalibacterium in PPI users compared to non-users microbiota in the total number, composition, and relative abun- reported elsewhere(46). A study conducted using healthy male dogs dance of species. demonstrated that decreased the Faecalibacterium The taxonomic level assessment revealed a lower relative count in healthy male dogs(47). In order to reduce the influence of abundance of the genus Faecalibacterium and a higher rela- PPI in the intestinal microbiota, subjects included in the present tive abundance of the family Clostridiaceae in the EE patients study had purportedly not used PPIs for at least 30 days preceding (FI­GURES 5 and 7). their participation. A recovery of intestinal microbiome 30 after Faecalibacterium prausnitzii is the only species that has been suspending the use of PPI has been described(48). However, further identified in the genus Faecalibacterium. F. prausnitzii is the main studies are still needed to better evaluate the changes caused by PPI representative of the phylum Firmicutes, class Clostridium, fam- in the intestinal microbiome. ily Ruminococcaceae. In humans, the genus Faecalibacterium is This study has some limitations. Firstly, based on the Lyon divided into two different phylogroups, although whether they Consensus, erosive esophagitis grade A and B are less accurate in the have different physiological functions is unknown(34). F. prausnitzii diagnosis of gastroesophageal reflux disease. Similar studies may is extremely sensitive to oxygen and is difficult to cultivate, even be performed in non-erosive reflux disease and compared with the in anaerobic conditions(34). F. prausnitzii represents approximately erosive form. Secondly, dietary surveys were not performed. Such 5% of the total fecal microbiota in healthy adults, and may reach surveys can be useful in evaluating the intestinal microbiota, as 15% in some individuals(35). The abundance and ubiquity of F. the diet has an influence on bacterial composition(49,50). The study prausnitzii suggest that this is a functionally important member participants lived in the same region, which may have reduced the of the microbiota, with a possible impact on the physiology and variation in the diets. Besides, the frequency and consistency of health of the host. Changes in the abundance of this bacterium stools of the participants, which may have an impact in the com- have already been widely described in different intestinal and meta- position of the intestinal microbiota, were not evaluated(51). There bolic diseases in humans(34). The beneficial effects of F. prausnitzii was also no metabolomics assessment of the samples. In addition, reflect its ability to produce butyrate, which positively modulates the sample size was small, which prevented extrapolation of the the intestinal immune system, oxidative stress, and the metabolism findings to other populations. of colonocytes(36-38). F. prausnitzii was reported to secrete anti- inflammatory compounds, such as salicylic acid(39). In a recent study, CONCLUSION seven peptides present in the supernatant from F. prausnitzii cultures were derived from a single anti-microbial inflammatory molecule, In conclusion, reduced abundance of the genus Faecalibacte- a 15 kDa protein, capable of blocking the nuclear factor-kappa B rium and greater abundance of the family Clostridiaceae may con- pathway in intestinal epithelial cells(39). Another study described tribute to the development of erosive esophagitis. Further studies that patients with reduced abundance of F. prausnitzii displayed are needed to confirm these findings and the importance of using higher serum levels of interleukin 8. The authors concluded that a therapeutic strategy for this clinical condition. alterations in the microbial composition are associated with an increase in intestinal permeability and increased plasma levels of ACKNOWLEDGEMENTS pro-inflammatory cytokines(40). Others reported that administra- tion of F. prausnitzii restored serotonin levels in the colon of rats We thank Instituto de Medicina Tropical de São Paulo for its with low-grade chronic (41). Although serotonin is contribution to the metagenomic laboratory analysis. We also not a direct marker of motility, it stimulates peristalsis, secretion, thank Alex Jones Flores Cassenote, Universidade de São Paulo, vasodilation, and sensory signaling in the intestine, and directly for statistical advice. and indirectly regulates intestinal motility(42). Finally, we observed increased abundance of the Clostridiaceae. Authors’ contribution Greater abundance of Clostridiaceae has already been described Baima DC: conceptualization, methodology, validation, investi- in other pro-inflammatory pathological contexts. In one study, gation, data curation, writing – original draft. Carvalho NS: writing a greater abundance of Clostridiaceae was observed in patients – review & editing, visualization. Barbuti RC: conceptualization, with inflammatory bowel disease and in patients with rheumatoid supervision, writing – review & editing, project administration. arthritis(43). An increase in the abundance of Clostridiaceae was Navarro-Rodriguez T: conceptualization, methodology, validation, also observed in infants with food (44). writing – original draft, writing – review & editing, supervision, The collective results of our study indicate differences in the project administration. All authors approved the final version of microbiota associated with the generation of a pro-inflammatory this manuscript bowel environment, with direct and indirect effects on the function of the digestive tract, including its upper segment. An important Orcid consideration is the possibility that the reduction in the abundance Diego Cardoso Baima: 0000-0002-6430-5431. of Faecalibacterium may be due to a possible prior use of PPIs by Nayara Salgado Carvalho: 0000-0001-9167-8957. patients with EE. The use of PPIs can be associated with the re- Ricardo Correa Barbuti: 0000-0002-1910-119X. duced abundance of Faecalibacterium in the intestinal microbiome. Tomas Navarro-Rodriguez: 0000-0002-3458-699X.

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Baima DC, Carvalho NS, Barbuti RC, Navarro-Rodriguez T. Avaliação da microbiota intestinal em adultos com esofagite erosiva. Arq Gastroenterol. 2021;58(2):168-74. RESUMO – Contexto – A doença do refluxo gastroesofágico (DRGE) é uma das enfermidades mais comuns na prática clínica e possui fisiopatologia multifatorial. Disbiose da microbiota intestinal pode ter influência em mecanismos envolvidos nesta doença, como mudanças nos padrões motores gastrointestinais, elevação da pressão intra-abdominal e aumento da frequência de relaxamentos transitórios do esfíncter esofágico inferior. Contudo, a avaliação da microbiota intestinal, neste contexto, ainda é pouco documentada. Objetivo – Este estudo avaliou a microbiota bacteriana intestinal, em indivíduos com doença do refluxo gastroesofágico erosivo e em indivíduos saudáveis, utilizando técnicas de metagenômica. Métodos – Estudo incluiu amostras fecais de 22 adultos, com idades entre 18 e 60 anos: 11 com esofagite erosiva (oito homens e três mulheres) e 11 controles saudáveis (dez homens e uma mulher). Os pacientes foram orientados a coletar e armazenar o material fecal em tubo contendo solução de guanidina. O DNA da microbiota foi extraído das amostras de fezes e amplificação por PCR foi realizada usando iniciadores para a região V4 do gene 16S rRNA. Osamplicons foram seqüenciados usando a plataforma Ion PGM Torrent e os dados foram analisados usando o software QIIME™ versão 1.8 (Quantitative Insights Into Microbial Ecology). Análise de estatística foi realizada utilizando-se o teste não paramétrico de Mann-Whitney e o teste ANOSIM, método não paramétrico baseado em matriz de distância. Resultados – Os índices de alfa-diversidade e beta-diversidade foram semelhantes entre os dois grupos, sem diferença estatisticamente significante. Não houve diferença estatisticamente significante no nível de filo, classe e ordem. Entretanto, observou-se diferença estatisticamente significante na abundância da famíliaClostridiaceae (0,3% vs 2,0%, P=0,032) e no gênero Faecaliumbacterium (10,5% vs 4,5%, P=0,045) entre controles saudáveis e pacientes com DRGE erosiva, respectivamente. Conclusão – Os achados sugerem que menor abundância do gênero Faecaliumbacterium e maior abundância da família Clostridiaceae, nos pacientes com DRGE, podem influenciar na fisiopatologia desta doença. Palavras-chave – Esofagite erosiva; microbiota intestinal; metagenômica; Faecalibacterium; Clostridiaceae.

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